Revisiting the Role of VraTSR in Staphylococcus aureus Response to Cell Wall-Targeting Antibiotics

ABSTRACT Exposure of Staphylococcus aureus to cell wall inhibitors leads to the activation of the VraTSR three-component sensory regulatory system. This system is composed of VraS, a membrane histidine kinase; VraR, its cognate response regulator, and VraT, a protein required for the full activity of VraTSR. The exact function of VraT remains mostly uncharacterized, although it has been proposed to detect the unknown stimulus sensed by the VraTSR system. Here, we elucidate the topology of VraT, showing that its C-terminal domain is extracellular. We also demonstrate that the signal sensed by VraTSR is not an intermediate in the peptidoglycan synthesis pathway, as previously suggested. Instead, the specific inhibition of the penicillin-binding protein (PBP)2 leads to strong activation of the system. IMPORTANCE The Gram-positive bacterial pathogen Staphylococcus aureus is currently the second most frequent cause of global deaths associated with antibiotic resistance. Its response to cell wall-targeting antibiotics requires the VraTSR three-component system, which senses cell wall damage. Here, we show that the signal sensed by VraTSR is not an intermediate in the peptidoglycan synthesis pathway, as previously suggested. Instead, the specific inhibition of the penicillin-binding protein (PBP)2, the major peptidoglycan synthase in S. aureus, leads to strong activation of the system. Identifying the exact cell wall damage signal is key to fully understanding the response of S. aureus to cell wall-targeting antibiotics.

To construct the vraTSR promoter fusion, an 814-bp DNA fragment containing the vraTSR promoter region was amplified from COL genomic DNA using primers PvraSR_P1_KpnI and PvraSR_P2_New_XhoI, digested with KpnI and XhoI, and cloned into KpnI/XhoI digested pFAST3, upstream of sfgfp-p7, resulting in plasmid pPvra-pFAST3, confirmed by DNA sequencing. pPvrap-FAST3 was electroporated into the S. aureus RN4220 strain and integrated into the chromosome at the vraTSR promoter site by homologous recombination, as confirmed by PCR and sequencing; the resulting strain was named RN Pvra-sGFP. Strain COL Pvra-sGFP was constructed by transducing the integrated plasmid pPvra-pFAST3 from RN Pvra-sGFP into COL using phage80α, as previously described 1 .
To construct the S. aureus pbpB null mutant, lacking PBP2, we amplified 1Kb DNA fragments from S. aureus COL genomic DNA corresponding to the upstream (primers PBP2_KO-P1 and PBP2_KO-P2) and downstream (primers PBP2_KO-P13 and PBP2_KO-P4) regions of the pbpB gene. The resulting PCR products were joined by overlap PCR using primers PBP2_KO-P1 and PBP2_KO-P4. The overlap PCR product was digested with EcoRI and BamHI and cloned into the thermosensitive plasmid pMAD 2 , producing plasmid ppbpB.
The plasmid was sequenced and introduced into RN4220 by electroporation 3 .
Following electroporation, the plasmid was transduced into COL using phage 80 as previously described 1 . Insertion and excision of ppbpB was performed as previously described 2 , except the integration steps were performed at 37 o C instead of 43 o C, resulting in strain COLpbpB. Deletion of the target gene was verified by PCR and resulting strains were verified by whole genome sequencing.
To investigate the mechanism behind PBP2-dependent VraTSR activation, COLΔpbpB_Pvra-sGFP was complemented with plasmids encoding different alleles of PBP2. For that, a 2214-bp fragment encompassing the wild type pbpB allele was amplified using primers PBP2muts_P1 and PBP2muts_P2 from S. aureus COL genomic DNA. The same pair of primers was used to amplify a fragment, coding for the transglycosylase-inactive PBP2 E114Q from COLTG42 4 genomic DNA. These fragments were introduced into SmaI-digested pCNX using a Gibson assembly cloning kit (NEB), originating plasmids pPBP2 and pPBP2TG.
To localize VraT and VraS, an N-terminal fusion of VraT to the P7 variant of superfast GFP (sfGFP) 5   , oxacillin (+Oxa, 800 µg mL -1 ) and vancomycin (+Van, 2 µg mL -1 ). Incubation in TSB without antibiotics was used as negative control. In the presence of all antibiotics tested, septal enrichment of PBP2 is lost or decreased and the protein becomes dispersed over the membrane. Scale bar = 1µm Supplemental Tables   Supplementary Table 1